Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes

Published: April 30, 2024

Abstract

Source: Santana-Magal, N., et al. Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes. J. Vis. Exp. (2018)

This video demonstrates a protocol for assessing the activation of dendritic cells using immunoglobulin G (IgG)-coated tumor cells. Dendritic cells internalize the IgG-coated tumor cells, process the tumor antigens, and present them on the surface through MHC-II molecules, along with the co-expression of the costimulatory molecule CD86. These dendritic cells then undergo MHC-II and CD86-targeted immunostaining and flow cytometry analysis to identify their activation state.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Isolation of Tumor-Associated Monocyte-Derived Dendritic Cells In a laminar flow hood, remove tumors from CO2 euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).   NOTE: Shaving the mice and spraying them with 70% ethanol before removing the tumors is highly recommend…

Divulgaciones

The authors have nothing to disclose.

Materials

Ficoll-Paque PREMIUM GE-Healthcare 17-5442-02
OptiPrep StemCell Technologies 7820
CD45 MicroBeads Miltenyi 130-052-301
EasySep Monocyte Isolation Kit StemCell Technologies 19861
Collagenase IV Sigma C9697-50MG Test each lot for endotoxin
DNase I Sigma DN25-10MG
HBSS ThermoFisher 14025092
FBS ThermoFisher 16140071 Test each lot for endotoxin
PE-CD11c Biolegend 117307
APC-CD11b Biolegend 101211
Brilliant Violet 650 MHCII Biolegend 107641
AF48- CD86 Biolegend 105017
APC/Cy7-Ly-C6 Biolegend 108423
PE/Cy7-CD15 Biolegend 135523

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Flow Cytometry-Based Analysis of Dendritic Cell Activation Using Immune Complexes. J. Vis. Exp. (Pending Publication), e22122, doi: (2024).

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